Multiple reaction monitoring (MRM), also known as selected reaction monitoring (SRM) mass spectrometry (MS), allows for fast and reproducible measurement of tens to hundreds of proteins in complex biological samples such as bio-fluids, tissues, and cultured cells. There is tremendous interest in applying the technology to develop blood-based clinical tests for the diagnosis, prognosis or treatment stratification of various diseases. Due to the high complexity of the human blood proteome, proteomic analysis of blood samples typically consists of multiple experimental steps and is prone to variation (FIG. 1A). In addition, changes in laboratory conditions (e.g., operators, instruments, reagents) are expected during routine laboratory operations in clinical testing. Therefore, controlling analytical variability to satisfy rigorous quality control requirements for blood-based clinical testing using MRM-MS platforms has been challenging.
The principle of stable isotope labeling (SIL) is currently used in MS-based quantitative proteomics to control experimental variability. Protein abundance is measured by comparing MS signal intensities of endogenous peptides with those of their corresponding stable isotope-labeled internal standard (SIS) peptides. Three SIL approaches are potentially suitable for clinical testing (FIG. 1B). The first approach utilizes SIS peptides for protein quantification (SISQuan) and is the simplest one for implementation. SIS peptides are synthesized, optimized for MS analysis and spiked into samples before or after protein digestion to control variation in post-digestion procedures. However, variation occurring before or during digestion is not controlled. The second approach spikes full-length SIS proteins into samples before any analytical procedure takes place. While this approach offers the best control of analytical variability, it is applicable only to soluble proteins. Quality control of the production, the storage, etc., of SIS proteins as standards is challenging for routine laboratory operations. The third approach spikes either artificial or truncated SIS proteins into samples before protein digestion. It controls most variation in protein digestion and variation in subsequent procedures. However, it cannot control variation occurring before digestion and faces similar implementation challenges as the second approach. None of the above SIL approaches can control pre-analytical variability associated with sample collection and handling.
Thus, there is a need for a simple and robust method that provides sufficient control of pre-analytical and analytical variability for routine clinical testing on MS-based proteomics platforms. The present invention addresses that need.